Project Summary. Over 1.25 million emergency department visits occurred in 2011 due to the abuse of illicit drugs and prescription opioids resulting in an estimated cost to the United States of $37 billion/year in healthcare costs and $271.5 billion/year overall when crime and lost productivity are included. The majority of these cases were due to cocaine (40%), heroin (21%), (meth)amphetamines (13%), and PCP (6%) with numbers rising in recent years for fentanyl, fentanyl analogues, and drugs (e.g. cocaine) laced with fentanyl. For this reason, the federal government considers the drug abuse (opioid) epidemic to be a national emergency. Currently, opioid overdose can be treated with the opioid receptor antagonist Naloxone via a pharmacodynamic (PD) strategy. However, with the rising abuse of fentanyl, Naloxone is becoming much less effective and multiple doses are often required. Furthermore, there are no current pharmacotherapies for intoxication with stimulants (e.g. methamphetamine, cocaine, or mephedrone) or for hallucinogens (e.g. ketamine or PCP) and ER doctors deliver treatments the only target the patient's symptoms. Accordingly, the development of new pharmacotherapies for the treatment of intoxication with the full range of drugs of abuse (opioids, stimulants, hallucinogens) is urgently needed. In contrast to PD strategies which target the opioid receptor, pharmacokinetic strategies target the drug itself for sequestration and/or catalytic destruction. Cucurbit[n]urils (CB[n]) are molecular container compounds that possess exceptionally tight binding (Ka routinely in the 106 ? 1012 M-1 range) toward hydrophobic cations in water which renders them a prime platform to create new in vivo sequestration agents for drugs of abuse. In key preliminary results we have shown that (acyclic) CB[n] bind strongly (Ka > 106 M-1) toward cocaine, methamphetamine, ketamine, PCP, fentanyl, and oxycodone in vitro and that Calabadion 2 is capable of reducing the hyper-locomotor activity observed upon treatment of rats with cocaine or methamphetamine. We propose to systematically vary the structure of the (acyclic) CB[n] receptors to optimize the binding affinity toward specific drugs of abuse. For example, Calabadions will be prepared with: 1) covalent caps to provide a 2 bind site for pendant functionality, 2) strategically placed SO3- groups to enhance ion-ion and ion-dipole interactions, 3) enhanced cavity diameters, and 4) extended aromatic walls. New sequestration agents will be subjected to a series of in vitro toxicology (Cytotoxicity: MTS metabolic and AK release cell death; Cardiotoxicity: hERG ion channel inhibition; Mutagenicity: Ames test) and in vivo maximum tolerated dose studies to establish their biocompatibility. Finally, hyperlocomotion assays will be used to determine the in vivo efficacy of the most promising sequestration agent / drug pairs. Upon completion of the proposed work we expect at least one sequestration agent will be nominated to continue toward a US FDA investigational new drug status.